Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy

Zhang, Xin; Tamaki, Hajime; Kikukawa, Takashi; Fujiwara, Toshimichi; Matsuki, Yoh

doi:https://doi.org/10.1016/j.bpc.2024.107329

Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy

Zhang, Xin; Tamaki, Hajime; Kikukawa, Takashi et al.

Biophysical Chemistry, 2024, 315, 107329

アクセス数:573件（2026-02-15 21:10 集計）

固定URL: https://hdl.handle.net/11094/98585

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タイトル	Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy
著者	著者名 Zhang, Xin
	researchmap permalink htamaki547 htamaki547 著者名 Tamaki, Hajime
	NRID 1000020281842 1000020281842 著者名 Kikukawa, Takashi
	NRID 1000020242381 1000020242381 researchmap permalink read0009115 read0009115 Scopus Author ID 7403542603 7403542603 著者名 Fujiwara, Toshimichi
	NRID 1000070551498 1000070551498 researchmap permalink yohmatsuki yohmatsuki Scopus Author ID 7006127172 7006127172 著者名 Matsuki, Yoh
キーワード等	Chloride ion transport
	Dynamic nuclear polarization
	Flash-photolysis
	Halorhodopsin
	Solid-state NMR spectroscopy
	Trimer assembly
内容	Zhang X., Tamaki H., Kikukawa T., et al. Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy. Biophysical Chemistry 315, 107329 (2024); https://doi.org/10.1016/j.bpc.2024.107329.
抄録	Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl− inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl− uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl− uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl−-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl− depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl− uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl− uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl−-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl− entrance path on the extracellular surface of the protein and is crucial to the Cl− uptake process.
出版者	Elsevier B.V.
収録物名	Biophysical Chemistry
巻(号)	315
ページ	107329
刊行年月	2024-12-01
言語	英語
ハンドルURL	https://hdl.handle.net/11094/98585
PISSN	03014622
eISSN	18734200
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1016/j.bpc.2024.107329
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCTERMS.bibliographicCitation	Biophysical Chemistry. 2024, 315, p. 107329
DC.title	Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy
DC.creator	Zhang, Xin
	Tamaki, Hajime
	Kikukawa, Takashi
	Fujiwara, Toshimichi
	Matsuki, Yoh
DC.publisher	Elsevier B.V.
DC.language' scheme='DCTERMS.RFC1766	英語
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2024-12-01
DC.identifier	https://doi.org/10.1016/j.bpc.2024.107329
DC.subject	Chloride ion transport
	Dynamic nuclear polarization
	Flash-photolysis
	Halorhodopsin
	Solid-state NMR spectroscopy
	Trimer assembly
DC.description	Zhang X., Tamaki H., Kikukawa T., et al. Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy. Biophysical Chemistry 315, 107329 (2024); https://doi.org/10.1016/j.bpc.2024.107329.
DCTERMS.abstract	Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl− inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl− uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl− uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl−-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl− depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl− uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl− uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl−-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl− entrance path on the extracellular surface of the protein and is crucial to the Cl− uptake process.
DC.rights	This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
DC.format	98585
citation_title	Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy
citation_author	Zhang, Xin
	Tamaki, Hajime
	Kikukawa, Takashi
	Fujiwara, Toshimichi
	Matsuki, Yoh
citation_publisher	Elsevier B.V.
citation_language	英語
citation_date	2024-12-01
citation_journal_title	Biophysical Chemistry
citation_volume	315
citation_firstpage	107329
citation_doi	https://doi.org/10.1016/j.bpc.2024.107329
citation_public_url	https://hdl.handle.net/11094/98585

Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy

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